Protein folding is a central life process. Errors in folding are the cause of many diseases and an understanding of protein folding is needed for wide ranging fields such as protein design, evolution, and drug design. Consequently, massive efforts have been directed at various aspects of folding. In the quest to understand protein folding, however, membrane proteins have been largely ignored. Membrane protein folding must be brought up to speed. We propose to learn more about the unfolded state in bilayers, the process of membrane protein folding, and how the final structure is defined. We will emerge from the proposed studies with new technologies for membrane protein folding studies, and with a much improved picture of how membrane proteins fold in true bilayer environments. The specific aims are: Aim I. (Folded state) Investigate the role of H-bonds in transmembrane helix bending. In the membrane where H-bonds can be very strong, mechanisms exist to allow transmembrane helices to bend. Helix bending is a fundamental feature of membrane protein structure and dynamics that must be understood. To investigate bending mechanisms we will employ a new method we have developed to measure backbone H-bond energies and observe how they correlate with sequence and structural changes. Aim II. (Folded state) How do changes in bilayer properties affect membrane protein stability? Lipid composition varies in different membrane domains, during signaling and as the result of disease. These changes can have profound effects on membrane protein function. Yet there are still no quantitative studies of bilayer composition effects on the stability of helical membrane proteins. We now have a method to attack this problem and will seek to fill in the gap in our knowledge. Aim III. (Folding) How do membrane proteins find the native state? The nature of the search toward the folded state remains largely uncharacterized for helical membrane proteins. We will characterize the folding transition state of a helical membrane protein in micelles for the first time, and develop methods for characterizing the folding process in bilayers. Aim IV. (Unfolded State) The structure of the unfolded state in membranes. The nature of the unfolded state of membrane proteins in bilayers is largely unknown. We now have a method for trapping the unfolded state in bilayers. We will characterize the structure of this unfolded state using EPR methods, thereby helping to complete a basic picture of membrane protein folding.